Coronaviruses(CoVs)are important infectious pathogens that are associated closely with respiratory and enteric diseases in humans and animals(Perlman and Netland, 2009; Belouzard et al., 2012; Li, 2013). CoVs have a single-strand positive sense RNA genome and consist of four groups: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus(Table 1).
Genus Species Receptor, co-receptor Alphacoronavirus HCoV-229E hAPN HCoV-NL63 ACE2 TGEV pAPN, Neu5Ac, Neu5Gc PEDV pAPN, Neu5Ac Betacoronavirus SARS-CoV ACE2 MHV CEACAM1 BCoV Neu5, 9Ac2 HCoV-OC43 Neu5, 9Ac2 Gammacoronavirus IBV Neu5Ac Deltacoronavirus Bulbul coronavirus HKU11 Unknown Thrush coronavirus HKU12 Munia coronavirus HKU13
Table 1. Coronavirus genera, species, and receptors.
Viral entry into cells is highly dependent on the interaction between viral particles and the host cells. CoVs use a variety of cellular receptors and co-receptors, including proteins and sugars, to facilitate their entry into cells. Infection begins with an interaction between the virus and its specific receptors(Li, 2015). Severe acute respiratory syndrome coronavirus(SARS-CoV)of group beta uses a type I integral membrane protein, angiotensin-converting enzyme 2(ACE2), as a receptor(Li et al., 2005). There is no structural homology between SARS-CoV and the Human coronavirus NL63(HCoV-NL63) receptor-binding domain(RBD), but they recognize the same receptor, ACE2(Wu et al., 2011). Although the RBD crystal structures of transmissible gastroenteritis virus(TGEV) and HCoV-NL63 are similar, they use different receptors(Reguera et al., 2012). Aminopeptidase N(APN), also known as CD13, is a type II transmembrane protein(Tusell et al., 2007). TGEV and porcine epidemic diarrhea virus(PEDV)infect cells through interaction with porcine(p)APN(Li et al., 2007; Nam and Lee, 2010). Human(h)APN is a receptor for HCoV-229E(Belouzard et al., 2012). In the same beta group, the receptors for mouse hepatitis virus(MHV) and bovine coronavirus(BCoV)are carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and a sugar, respectively, despite their high sequence homology(Peng et al., 2011; Peng et al., 2012). CEACAM1, the first identified coronavirus receptor(Dveksler et al., 1991), is a type I transmembrane multifunctional protein of the immunoglobulin superfamily(de Haan et al., 2005).
Cell entry and interspecies transmission of CoVs are mediated by the spike protein(S). CoV S is a class I fusion protein(Bosch et al., 2003). In CoVs, S is a significant surface protein and plays an important role in mediating infection of virions(Schwegmann-Wessels et al., 2009). S is also responsible for receptor binding and the fusion of viral and cellular membranes. All CoV S share the same functional component in two domains: an N-terminal domain called S1 that is responsible for receptor binding, and a C-terminal S2 domain that is responsible for fusion. S1 contains two subdomains, an N-terminal domain and a C-terminal domain(Figure 1A). Both function as RBDs and bind a variety of proteins and sugars(Belouzard et al., 2012).
Figure 1. S proteins from coronaviruses of different groups. (A) Domain structure of the S protein, including the S1 and S2 regions, transmembrane domain (TM), and cytoplasmic tail (Cyto). (B) Amino acid sequence identities of S proteins from coronaviruses of different groups. The GenBank accession numbers are as follows: ABD72982.1 for SARS-CoV-S, AAR92025.1 for MHV-S, NP_073551.1 for HCoV-229E-S, ABG89335.1 for TGEV-S, NP_598310.1 for PEDV-S, AAT84362.1 for HCoV-OC43-S, ABM66810.1 for BCoV-S, and NP_040831.1 for IBV-S. (C) Amino acid sequence identities of S1 proteins from coronaviruses of different groups.
Because some CoVs have strong pathogenicity, the lentiviral pseudotype system is a reliable tool to study the proteins of highly pathogenic viruses under conventional biosafety conditions. HIV-Luc is an HIV-1 based lentiviral vector bearing the luciferase reporter gene and has been used in the production of pseudotyped viruses(Kang et al., 2012; Lu et al., 2012; Tang et al., 2012). Pseudovirus entry efficiency is characterized based on the luciferase activity. Viral particles pseudotyped by various S proteins have been described for several viruses. In our study, we compared the efficiency of pseudotyped viruses with S proteins from different groups of CoVs. Furthermore, the cell tropisms of TGEV and PEDV were characterized by live and pseudotyped viruses.